GSM7710929: ALLO, replicate 1, scRNAseq; Mus musculus; RNA-Seq
The spleens were dissociated into single-cell splenocytes using fully frosted glass slides. The splenocytes were filtered through a 40 µm cell strainer (Falcon, Durham, NC), and preserved in cryomedia (90 % FBS + 10 % DMSO) for scRNA-seq analysis. Cell stocks were thawed in 37℃ 10% FBS/DMEM. The cells were washed twice with cold Ca2+- and Mg2+-free 0.04 % BSA/PBS at 300g for 5min at 4°C. Samples were gently resuspended in cold Ca2+- and Mg2+-free 0.04% BSA/PBS and then counted using a LUNA-FX7™ Automated Fluorescence Cell Counter (Logos biosystems) with AO/PI staining. Cell viability was further assessed using the Dead Cells Removal Kit (Miltenyi biotech, Cat no 130-090-101) and MS columns (Miltenyi biotech, cat no. 130-042-201) according to the manufacturer's instructions. According to the 10x Chromium Single Cell 5' v2 protocol (10x Genomics, document no. CG000331), the scRNA-seq libraries were prepared using the 10x Chromium controller and Next Gem Single Cell 5' Reagent v2 kits (10x Genomics, PN-1000244).